The long-term objective of this proposed research is to understand the genetic and biochemical factors involved in the patterning and maintenance of a stem cell niche. The proposed studies will test the hypothesis that the generation of a local paracrine signal, the phytohormone cytokinin, specifies cell identity and controls rates of cell division in the stem cell niche of the Arabidopsis thaliana shoot apical meristem (SAM). The anatomy of a stem cell niche is spatial and temporally defined, where domains of gene expression and cell activity form microenvironments that provide signals to regulate stem cell proliferation, differentiation, survival, and localization. Therefore, the goals of the specific aims proposed herein, are directed to understand the spatial and temporal regulation of cytokinin metabolism in the patterning and specification of stem cell identity and maintenance using the shoot apical meristem of Arabidopsis thaliana as a model system. The first two aims of this study will take advantage of the unique situation inherent to Arabidopsis that makes it a tractable system to study stem cell dynamics;the stem cell population of the SAM is exposed at the very tip of the shoot making it readily accessible to in vivo live imaging of stem cell populations by laser scanning confocal microscopy (LSCM). Specific aim 1 will map the cellular expression domains in vivo by LSCM live imaging of fluorescent reporter genes for the ISOPENTLYTRANSFERASE (IPT), LONELY GUY (LOG), and CYTOKININ OXIDASE (CKX) gene families in the stem cell niche of the SAM. The second aim will build upon the first, interrogating the dynamics of local cytokinin synthesis in the different domains of the SAM using chemical and transient genetic intervention techniques to temporally and spatially repress cytokinin synthesis to assess its function in specification of domain identity and cell proliferation. In specific aim 3 a yeast one-hybrid screen will be performed to identify novel transcription factors that regulate the expression of the cytokinin metabolic genes. The identification of domains of genes expression, cellular activity, and mechanisms that regulate the production a local paracrine within the stem cell niche will allow for the generation of more complete models of how cell proliferation, differentiation, survival, and specification of cell identity are control in the a stem cell niche. PUBLIC HEALTH RELEVANCE: Understanding how stem cell populations are maintained in tissues is a central question that needs to be answered if we are to realize the full therapeutic potential of stem cells in the treatment of human disease. Characterizing the cellular signals that maintain and regulate stem cell populations will allow for the development of new technologies to utilize stem cells in such applications as tissue and organ regeneration.